Radioactive benzodiazepine derivatives

ABSTRACT

The present invention relates to a novel radioactive benzodiazepine derivative represented by the formula: ##STR1## wherein R is hydrogen or a lower alkyl; X* is radioactive iodine or bromine; Y is hydrogen or a halogen; and Z is a halogen or nitro group; and the salt thereof. 
     The compound of the present invention enables to non-invasively detect the presence of benzodiazepine receptors in human or animal brains, other organs or tissues. Additionally, it enables to dynamically trace the change in receptor concentration either. It is very useful as a radioactive diagnostic agent for nuclear medicine in vitro or in vivo or as a radio-pharmaceutical.

The present invention relates to a novel radioactive benzodiazepinederivative represented by the formula (I): ##STR2## wherein R ishydrogen or a lower alkyl; X* is radioactive iodine or bromine; Y ishydrogen or a halogen and Z is a halogen or nitro group; and the saltthereof (hereinafter referred to as the compound of the presentinvention) as well as processes for producing the same.

The compound represented by the formula (I) is a novel compound notdisclosed in any literature and has a very high affinity tobenzodiazepine receptors. The compound is very useful as a radioactivediagnostic agent for nuclear medicine in vitro or in vivo or aradiopharmaceutical. Furthermore, the compound of the present inventionis very useful as a radioactive ligand in radio-immunoassay method, asit has a high affinity to benzodiazepine antibodies.

In recent years, it has been found that the amount of benzodiazepinereceptors changes in a certain brain disorder (for example, epilepsy,dementia, etc.). In the medical or pharmaceutical field, studies on arelationship between benzodiazepine receptors and various cerebraldiseases are of great interest. Under these circumstances, it has beenstrongly desired to develop diagnostics and radio-pharmaceuticals,targetting to benzodiazepine receptors.

As such an approach, compound [C-11]Ro 15-1788 labeled with positronemitter, C-11 has recently been developed and, benzodiazepine receptorsin the human brain have been investigated by the PET method [J.Psychiatr. Res., 19, 609 (1985); Radioisotopes, 34, 302 (1985)].

However, the aforesaid method using [C-11]Ro 15-1788 involves manyproblems in practical use. That is, cyclotron facilitates are requiredin the clinics because C-11 has an extremely short half life. Besides,the labeled compound must be synthesized in a short period of time, etc.

The present inventors have made extensive investigations, aiming atdeveloping benzodiazepine receptor diagnostics and radiopharmaceuticalslabeled with radioactive iodine or bromine. As a result, it has beenfound that the compound represented by the formula (I) defined above hasa very high affinity to benzodiazepine receptors and specifically bindsthereto. A combination system of a compound selected from the compoundsof the present invention with single photon emission computed tomography(SPECT) method solves the aforesaid problems PET method involves. Thistechnique gives not only easy determination or diagnosis ofbenzodiazepine receptors but also receptor imaging at several hoursafter administration, which cannot be obtained with [C-11]Ro 15-1788 dueto its super short half life. That is, the present inventors have foundthat the compound of the present invention represented by the formula(I) possesses excellent properties as benzodiazepine receptor targettingdiagnostics and radiopharmaceuticals from a practical viewpoint and haveaccomplished the present invention.

The methods for preparing the compound of the present invention aredescribed below.

The compound of the present invention represented by the formula (I)defined above can be produced according to either Process A or Process Bshown below.

[Process A]

The compound of the present invention represented by the formula (I)defined above can be obtained by an exchange reaction of abenzodiazepine derivative represented by the formula (II): ##STR3##wherein R, Y and Z are the same as defined above and X is iodine orbromine; with a radioactive metal iodide or metal bromide in a solvent,for example, acetonitrile, dimethyl sulfoxide, dimethylformamide,ethylene glycol, an ethereal derivative of ethylene glycol, an etherealderivative of diethylene glycol, hexamethylphosphorous triamide (HMPT),water or the like, generally at a reaction temperature of 50° to 180° C.followed by a conventional manner such as extraction with a solvent,etc.

[Process B]

An aminobenzodiazepine derivative represented by the formula (III):##STR4## wherein R, Y and Z are the same as defined above, is allowed totreat with an alkali metal nitrite in a solvent (e.g. tetrahydrofuran,dioxan, acetonitrile) in the presence of an acid (e.g. diluted sulfuricacid, an organic acid) to form a diazonium salt represented by theformula (IV): ##STR5## wherein R, Y and Z are the same as defined above,A⁻ is a halogen ion, or an anion having the formula: HSO₄ ⁻, theformula: ##STR6## or the formula R'B⁻ wherein R' is an alkyl, ahaloalkyl or an aryl which may be substituted, and B is a substituenthaving the formula SO₃ or CO₂.

Then, the diazonium salt represented by the formula (IV) is allowed totreat with a radioactive hydroiodic acid, hydrobromic acid, metal iodideor metal bromide, if necessary, in the presence of copper powders or acopper salt, generally at a temperature ranging from -5° to 30° C. Then,the product is isolated in a conventional manner such as extraction witha solvent, etc. to give the compound of the present inventionrepresented by the above formula (I).

The compound of the present invention obtained by either Process A orProcess B described above may be purified, if necessary or desired, in aconventional manner such as thin layer chromatography (TLC) orhigh-performance liquid chromatography (HPLC), etc.

In the present invention, the radioactive iodine includes I-123, I-125,I-131, I-132, etc. with I-123 being preferred. As the radioactivebromine, Br-75, Br-76, Br-77, Br-80, Br-82, etc. are exemplified. Theradioactive metal iodide or metal bromide means a metal salt of theabove radioactive iodine or bromine, and may be any of those capable ofproviding a radioactive I⁻ ion or Br⁻ ion. Specific examples thereofinclude sodium iodide, potassium iodide, lithium iodide, sodium bromide,potassium bromide, lithium bromide, etc.

As the halogen atom, fluorine, chlorine, bromine and iodine areexemplified.

The salt of the radioactive benzodiazepine derivative of the presentinvention refers to a pharmacologically acceptable salt such as salts ofthe radioactive benzodiazepine derivative with a mineral acid (e.g.hydrochloric acid, sulfuric acid) of those with an organic acid (e.g.acetic acid) etc.

After the radioactive benzodiazepine derivative or salt thereof obtainedby the present invention has been intravenously administered to thepatient, scintigram is taken with the passage of time or, radioactivityis measured by a probe method or, uptake of the compound into a specificorgan or tissue is measured by tomographic images obtained with a SPECTor PET camera, thereby enabling an easy and accurate diagnosis of theregional scope of focus and the degree of disease. Furthermore, thecompound of the present invention labeled with, for example, an atomsuch as I-125 or I-131, can be advantangeously used as a radioactiveligand for quantitatively analyzing the benzodiazepine derivative andits metabolite and for estimating the affinity, in radioactiveimmunoassay using the benzodiazepine antibody, in measurement of theamount of the benzodiazepine derivative in a body fluid sample (e.g.blood, urine, etc.) and in radioreceptor assay using the benzodiazepinereceptors.

The following Examples, Reference Examples and Test Examples serve togive specific illustrations of the practice of the present invention butthey are not intended any way to limit the scope of the presentinvention.

The following Reference Examples show the preparation of the compoundsused as a raw material for the compounds of the present invention.

REFERENCE EXAMPLE 1 Preparation of7-chloro-2,3-dihydro-5-(2-iodophenyl)-1-methyl-1H-1,4-benzodiazepine

After a mixture of 181 mg ofN'-(4-chlorophenyl)-N-(2-iodobenzoyl)-N'-methylethylenediamine, 925 mgof phosphorus pentoxide and 4 ml of phosphorus oxychloride had beenstirred at 115° C. to 120° C. for 4 hours, the reaction mixture wasallowed to cool and poured into ice water to decompose an excess of thereagents. After having been washed with ether, the aqueous phase wasalkalized with sodium carbonate and then extracted with ethyl acetate.After the organic phase had been washed with water, distilling off thesolvent therefrom gave 164 mg of7-chloro-2,3-dihydro-5-(2-iodophenyl)-1-methyl-1H-1,4-benzodiazepine.

Mass spectrum (70 eV) m/e: 396, 398 (M⁺).

¹ H-NMR (CDCl₃) δ (ppm): 2.9 (3H, s, CH₃), 3.6-3.9 (4H, m, --CH₂ CH₂--), 6.9-7.9 (7H, m, benzene ring H).

REFERENCE EXAMPLE 2 Preparation of7-chloro-1-methyl-5-(2-iodophenyl)-3H-1,4-benzodiazepin-2-one(2'-iododiazepam)

A THF solution of 158 mg7-chloro-2,3-dihydro-5-(2-iodophenyl)-1-methyl-1H-1,4-benzodiazepine anda sodium bicarbonate aqueous solution were simultaneously added by dropsto a THF solution of N-bromosuccinimide at room temperature. After themixture had been stirred at the same temperature for additional 30minutes, water was poured thereinto and the resulting mixture wasextracted with ethyl acetate. Purifying the crude product by silica gelcolumn chromatography gave 140 mg of 2'-iododiazepam.

Melting point: 174°-176° C.

Mass spectrum (70 eV) m/e: 410, 412 (M⁺).

¹ H-NMR (CDCl₃) δ (ppm): 3.4 (3H, s, CH₃), 3.8 (1H, d, CH), 4.7 (1H, d,CH), 6.9-7.9 (7H, m, benzene ring H).

REFERENCE EXAMPLE 3 Preparation of7-chloro-1-methyl-5-(2-bromophenyl)-3H-1,4-benzodiazepin-2-one(2'-bromodiazepam)

In the same procedure as in Reference Example 2, 170 mg of2'-bromodiazepam were obtained from 200 mg of7-chloro-2,3-dihydro-5-(2-bromophenyl)-1-methyl-1H-1,4-benzodiazepineobtained according to the Reference Example 1.

Melting point: 145°-146° C.

Mass spectrum (70 eV) m/e: 362, 364 (M⁺).

¹ H-NMR (CDCl₃) δ (ppm): 3.4 (3H, s, CH₃), 3.8 (1H, bd, CH), 4.8 (1H,bd, CH).

REFERENCE EXAMPLE 4 Preparation of7-nitro-1-methyl-5-(2-iodophenyl)-3H-1,4-benzodiazepin-2-one

A chromic acid solution was added by drops to an acetic acid solution of158 mg7-nitro-2,3-dihydro-5-(2-iodophenyl)-1-methyl-3H-1,4-benzodiazepine atroom temperature. After having been stirred at the same temperature foradditional one hour, the reaction mixture was poured into ice water andalkalized with aqueous ammonia. After the solvent had been distilledoff, purifying the resulting crude product by silica gel columnchromatography gave 70 mg of7-nitro-1-methyl-5-(2-iodophenyl)-3H-1,4-benzodiazepin-2-one.

Mass spectrum (70 eV) m/e: 421 (M⁺).

¹ H-NMR (CDCl₃) δ (ppm): 3.5 (3H, s, CH₃), 3.9 (1H, bd, CH), 4.7 (1H,bd, CH), 7.2-8.5 (7H, m, benzene ring H).

REFERENCE EXAMPLE 5 Preparation of7-chloro-1-methyl-5-(2-fluoro-4-iodophenyl)-3H-1,4-benzodiazepin-2-one(4'-iodofludiazepam)

To a mixture of 165 mg of 4'-aminofludiazepam, 0.8 ml of acetonitrileand a sodium nitrite aqueous solution was added 130 μl oftrifluoroacetic acid while cooling the mixture with ice water. Themixture was stirred at the same temperature for 20 minutes. An aqueoussolution of potassioum iodide was added to the obtained diazonium saltsolution followed by stirring at the same temperature for 2 hours. Aftercompletion of the reaction, the reaction mixture was extracted withchloroform. The solvent was distilled off to obtain the crude product.Purifying it by silica gel column chromatography gave 160 mg of4'-iodofludiazepam.

Mass spectrum (70 eV) m/e: 428, 430 (M⁺).

¹ H-NMR (CDCl₃) δ (ppm): 3.4 (3H, s, CH₃), 3.8 (1H, d, CH), 4.8 (1H, d,CH), 7.0-7.7 (6H, m, benzene ring H).

REFERENCE EXAMPLE 6 Preparation of7-chloro-1-methyl-5-(4-iodophenyl)-3H-1,4-benzodiazepin-2-one(4'-iododiazepam)

In the same procedure as in Reference Example 2, 160 mg of4'-iododiazepam were obtained from 180 mg of7-chloro-2,3-dihydro-5-(4-iodophenyl)-1-methyl-1H-1,4-benzodiazepine.

Mass spectrum (70 eV) m/e: 410, 412 (M⁺).

REFERENCE EXAMPLE 7 Preparation of7-chloro-1-methyl-5-(3-iodophenyl)-3H-1,4-benzodiazepin-2-one(3'-iododiazepam)

In the same procedure as in Reference Example 2, 140 mg of3'-iododiazepam were obtained from 182 mg of7-chloro-2,3-dihydro-5-(3-iodophenyl)-1-methyl-1H-1,4-benzodiazepine.

Mass spectrum (70 eV) m/e: 410, 412 (M⁺).

The following Examples show the preparation of the compounds of thepresent invention.

EXAMPLE 1 Preparation of 7-chloro-1-methyl-5-([¹²⁵I]-2-iodophenyl)-3H-1,4-benzodiazepin-2-one ([¹²⁵ I]-2'-iododiazepam)

To 10 μl of a DMF solution containing 5 μg of 2'-bromodiazepam wereadded 1-naphthalenesulfonic acid, copper sulfide and 1 mCi of Na¹²⁵ I.After having been heated to 100° C. for 1.5 hours, the reaction mixturewas allowed to cool. Purifying the obtained crude product by HPLC(column: Deverosil®ODS7) gave 0.7 mCi of [¹²⁵ I]-2'-iododiazepam. Thisproduct was identical with the product obtained in Reference Example 2in Rf values in TLC and Rt values in HPLC.

EXAMPLE 2 Preparation of 7-chloro-1-methyl-5-([⁸²Br]-2-bromophenyl-3H-1,4-benzodiazepin-2-one ([⁸² Br]-2'-bromodiazepam)

To 6 μg of 2'-iododiazepam obtained in Reference Example b 2 were addedto 10 μl of 50% aqueous solution of DMF, 1-napthalenesulfonic acid,copper sulfide and 2 mCi of Na⁸² Br. After having been heated at 100° C.for 2 hours, the reaction mixture was allowed to cool. Purifying theobtained crude product by HPLC gave 1 mCi of [⁸² Br]-2'-bromodiazepam.This product was identical with the product obtained in ReferenceExample 3 in Rf values in TLC and Rt values in HPLC.

EXAMPLE 3 Preparation of 7-nitro-1-methyl-5-([¹²⁵I]-2-iodophenyl)-3H-1,4-benzodiazepin-2-one

To 20 μl of DMF solution containing 10 μg of7-nitro-1-methyl-5-(2-iodophenyl)-3H-1,4-benzodiazepin-2-one obtained inReference Example 4 were added 1-naphthalenesulfonic acid, coppersulfide and 1 mCi of Na¹²⁵ I. After having been heated at 100° C. for 2hours, the reaction mixture was allowed to cool. Purifying the obtainedcrude product by HPLC (chloroform/acetone=9/1) gave 0.6 mCi of7-nitro-1-methyl-5-([¹²⁵ I]-2-iodophenyl)-3H-1,4-benzodiazepin-2-one.This product was identical with the product obtained in ReferenceExample 4 in Rf values in TLC.

EXAMPLE 4 Preparation of 7-chloro-1-methyl-5-(2-fluoro[¹²⁵I]-4-iodophenyl)-3H-1,4-benzodiazepin-2-one ([¹²⁵ I]-4'-iodofludiazepam)

In the same procedure as in Example 3, 0.3 mCi of [¹²⁵I]-4'-iodofludiazepam was obtained from 6 μg of 4'-iodofludiazepamobtained in Reference Example 5. The product was identical with theproduct obtained in Reference Example 5 in Rf values in TLC.

EXAMPLE 5 Preparation of 7-chloro-1-methyl-5-([¹²⁵I]-3-iodophenyl)-3H-1,4-benzodiazepin-2-one ([¹²⁵ I]-3'-iododiazepam)

In the same procedure as in Example 3, 0.3 mCi of [¹²⁵I]-3'-iododiazepam was obtained from 4 μg of 3'-iododiazepam obtained inReference Example 6. The product was identical with the product obtainedin Reference Example 6 in Rf values in TLC.

EXAMPLE 6 Preparation of 7-chloro-1-methyl-5-([¹²⁵I]-4-iodophenyl)-3H-1,4-benzodiazepin-2-one ([¹²⁵ ]-4'-iododiazepam)

In the same procedure as in Example 3, 0.5 mCi of [¹²⁵I]-4'-iododiazepam was obtained from 7 μg of 4'-iododiazepam obtained inReference Example 7. The product was identical with the product obtainedin Reference Example 7 in Rf values in TLC.

The following Test Example shows that the compound of the presentinvention has a very high affinity to benzodiazepine receptors.

TEST EXAMPLE

2'-Iododiazepam, Diazepam and Fludiazepam were screened as for dopaminereceptor binding affinity according to the method reported by Nakatsuka(Life Sciences 36 (2) 113-119, 1985). An aliquot of synaptosomalmembrane preparations was incubated at 40° C. for 15 min. with each ofthe unlabeled competing drugs (2'-Iododiazepam, Diazepam andFludiazepam) in different concentration and 3H-diazepam. The incubationwas terminated by adding ice-cold Tris-HCl buffer followed by a rapidfiltration through a Whatman GF/B filter. The bound 3H-diazepam retainedon the filter was extracted with ACS-II (Amersham) and counted. Allincubations were conducted in triplicate. Nonspecific binding wasdetermined in tubes containing diazepam. Specific binding was calculatedby subtracting the nonspecific binding from the total binding. IC₅₀values, the concentrations of the tested compounds that cause 50%inhibition of the specific 3H-diazepam were determined from thedisplacement curves obtained. The results were summarized in Table.

                  TABLE                                                           ______________________________________                                        Inhibitory Potency (Affinity for Benzodiazepine                               Receptors) of Benzodiazepine Derivatives for 3H--                             Diazepam to Rat Synaptosomal membranes                                                                            Relative                                  Compound   IC.sub.50 (M)                                                                              Ki (M)      Potency                                   ______________________________________                                        Diazepam    9.0 × 10.sup.-9                                                                      7.7 × 10.sup.-9                                                                    100                                       Fludiazepam                                                                               1.2 × 10.sup.-9                                                                      1.0 × 10.sup.-9                                                                    770                                       2'-Iododiazepam                                                                            2.5 × 10.sup.-10                                                                     2.1 × 10.sup.-10                                                                  3600                                      ______________________________________                                    

The compound of the present invention enables not only to non-invasivelydetect the presence of benzodiazepine receptors in human or animalbrains, other organs or tissues but also to dynamically trace the changein receptor concentration.

What is claimed is:
 1. A radioactive benzodiazepine derivativerepresented by the formula (I): ##STR7## wherein R is hydrogen or alower alkyl; X* is radioactive iodine or bromine; Y is hydrogen or ahalogen; and Z is a halogen or nitro group and the salt thereof.
 2. Aradioactive benzodiazepine derivative or the salt thereof according toclaim 1, wherein R is methyl.
 3. A radioactive benzodiazepine derivativeor the salt thereof according to claim 1, wherein the substituent X* isone member selected from the group of iodine isomers or bromine isomersconsisting of I-123, I-125, I-131, I-132, Br-75, Br-76, Br-77, Br-80 andBr-82.
 4. A radioactive benzodiazepine derivative or the salt thereofaccording to claim 3, wherein the substituent X* is I-125 or Br-82.
 5. Aradioactive benzodiazepine derivative or the salt thereof according toclaim 1, wherein Z is a halogen or nitro group at 7-position.
 6. Aradioactive benzodiazepine derivative or the salt thereof according toclaim 5, wherein Z is chlorine or nitro group.